Direct characterization of amyloidogenic oligomers by single-molecule fluorescence

Abstract

A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ؎ 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-␤ structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.amyloid aggregation ͉ amyloid oligomers ͉ two-color coincidence spectroscopy ͉ PI3-SH3 domain ͉ neurodegenerative diseases

Kinetics of the Interactions between Copper and Amyloid‐β with FAD Mutations and Phosphorylation at the N terminus

“…In order to unequivocally establish the oligomerisation mechanism in the presence of metal ions, Aβ peptides labelled with pairs of fluorophores would be necessary for the identification of co‐existing Aβ species. FRET or two‐colour coincidence detection at the single‐molecule level would allow direct measurement of time profiles for both copper‐bound Aβ monomer and copper‐bridged Aβ dimer 45, 46. However, mutations or post‐translational modifications on the N terminus of Aβ would not necessarily increase the reactivity of Aβ.…”

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation

“…3C), further reinforces the conclusion that a conformational change has occurred before their formation. This antibody is likely to recognize conformational characteristics of the intermediate species, perhaps because of the exposure of hydrophobic side chains (28,29) or the adoption of a different conformational form (30). The most protected residues in the O agg species are also the most protected ones in the F agg species, implying that these intermediates are likely to be species that progress to fibril formation.…”

Direct observation of multiple misfolding pathways in a single prion protein molecule

“…However, SM spectroscopy has not yet been widely applied to characterize protein misfolding. Misfolding has been observed in a few protein constructs using both force (5-7) and fluorescence spectroscopies (8), and the formation and growth of aggregates has been monitored with fluorescence spectroscopy (9)(10)(11), but the network of pathways available for misfolding has only begun to be mapped out in any detail (12) and not yet for any disease-related protein.…”